PROJECT SUMMARY Heart failure remains a leading cause of morbidity and mortality worldwide. A major issue in the setting of heart failure is loss of cardiomyocytes, and the inability of adult mammalian cardiomyocytes to replace themselves by cell cycle re-entry and cell division. In contrast, newts, zebrafish and neonatal mammalian cardiomyocytes can undergo mitotic cell division to regenerate the heart. Previous studies aimed at provoking cell cycle re- entry and proliferation of adult mammalian cardiomyocytes have met with partial success. Recalcitrance of adult cardiomyocytes to efficiently undergo proliferative cell division reflects epigenetic and transcriptional programs that dictate multiple properties of the adult cardiomyocyte state that provide barriers to their proliferative ability. These barriers include: activation of cell cycle inhibitors; repression of cell cycle activators; a metabolic state geared toward availability of relatively high oxygen levels with high numbers of mitochondria, abundant and highly organized myofibrillar structure, and high levels of binucleation. Thus, alteration of a signaling pathway or overexpression of a single cell cycle regulator may not be able to efficiently overcome all these obstacles. Instead, promoting efficient proliferation of adult cardiomyocytes is likely to require a multi- pronged approach, where each of these obstacles is overcome. Although we know much about epigenetic and transcriptional programs regulating cardiomyocyte development, our knowledge concerning epigenetic and transcriptional programs regulating the complex transitions from the fetal to adult cardiomyocyte state is limited. A comprehensive in depth understanding of these programs will give insight into mechanisms by which we can overcome multiple barriers within adult cardiomyocytes to promote cell cycle re-entry. In the proposed studies, we will examine cardiomyocyte cell cycle regulation by a key epigenetic regulator, Dot1L, identify transcription factor codes driving distinct states of cardiomyocyte cell cycle, examine effects of key metabolic transcription factors, HIFs, on adult cardiomyocyte proliferation, and investigate the potential role of atypical E2F factors on cardiomyocyte binucleation and proliferation. Results of these studies will be groundbreaking and be of future impact in that we hope to provide a roadmap of specific nodal points that can be targeted to allow the adult cardiomyocyte to undergo productive and regulated proliferation, thus paving the way for regenerative therapies for the heart. .